In order to solve a problem associated with pesticide residues that are contained in commercial vegetables and fruits, it is necessary to develop a method to screen pesticide residues rapidly and thus to invent a disposable, cheap bioassay kit, which can be used to detect pesticide residues immediately. Till recently, the major pesticides widely used in agricultural fields are organic phosphorus compounds and carbarmates; these pesticides can be detected by using a bioassay kit which is sensitive to the pesticides.
Enzyme immobilization is a critical technique for the development of a bioassay kit. Compared with immobilized enzymes in a “solution” state, immobilized enzymes in a “dry” state can reduce the decreasing rate of enzyme activity. This is highly advantageous to extend the preservative time of the bioassay kit. However, after the enzyme is immobilized and adsorbed onto the surface of a carrier, the activity of the enzyme declines to approximately one tenth of total activity of the original enzyme. Regarding the pesticides-detecting bioassay kits, the enzyme-immobilized carrier plays an influential role in the calorimetric process of pesticide residues screening when the Ellman test is used. The examination for color change on the enzyme-immobilized carrier by human eyes will provide a simple indication of enzyme inhibition or being free of inhibition due to the presence of pesticides or not. Once the enzyme-immobilized carrier is dipped into the aqueous substrate solution, elution of the enzyme or/and colorimetric products may occur, resulting in the reduction of detection sensitivity of the chromaticity by human eyes. There are a variety of methods for the immobilization of the enzyme, including physical adsorption, ion binding, covalent binding, cross-linking, entrapments, etc. According to U.S. Pat. No. 5,624,831, gelatin and trehalose can be used to form a stable film through a synergistic effect of these two materials and then an enzyme can be successfully entrapped into the stable film. The enzyme can maintain its activity at 100% and can be preserved up to 31 days under a dry condition and in a temperature of 4° C., room temperature, or even 50° C. Nevertheless, the enzyme immobilized by this method may reduce its activity to 70% after preserving 31 days under a dry condition and in temperature of 37° C.